Digital Perspectives in History

This article outlines the state of digital perspectives in historical research, some of the methods and tools in use by digital historians, and the possible or even necessary steps in the future development of the digital approach. We begin by describing three main computational approaches: digital databases and repositories, network analysis, and Machine Learning. We also address data models and ontologies in the larger context of the demand for sustainability and linked research data. The section is followed by a discussion of the (much needed) standards and policies concerning data quality and transparency. We conclude with a consideration of future scenarios and challenges for computational research

Upgrading LMSs: Understanding Faculty Perceptions and Use for Improved Implementation

Adventist schools are in a constantly changing academic environment and are under consistent pressure to implement the latest technologies, such as Learning Management Systems (LMSs). This research study seeks to provide a better understanding of faculty perceptions of their LMS. We explore factors leading to successful faculty use of LMSs

Small substrate, big surprise: fold, function and phylogeny of dihydroxyacetone kinases

Abstract.: Dihydroxyacetone (Dha) kinases are a family of sequence-conserved enzymes which utilize either ATP (in animals, plants and eubacteria) or phosphoenolpyruvate (PEP, in eubacteria) as their source of high-energy phosphate. The kinases consist of two domains/subunits: DhaK, which binds Dha covalently in hemiaminal linkage to the Nε2 of a histidine, and DhaL, an eight-helix barrel that contains the nucleotide-binding site. The PEP-dependent kinases comprise a third subunit, DhaM, which rephosphorylates in situ the firmly bound ADP cofactor. DhaM serves as the shuttle for the transfer of phosphate from the bacterial PEP: carbohydrate phosphotransferase system (PTS) to the Dha kinase. The DhaL and DhaK subunits of the PEP-dependent Escherichia coli kinase act as coactivator and corepressor of DhaR, a transcription factor from the AAA+ family of enhancerbinding proteins. In Gram-positive bacteria genes for homologs of DhaK and DhaL occur in operons for putative transcription factors of the TetR and DeoR families. Proteins with the Dha kinase fold can be classified into three families according to phylogeny and function: Dha kinases, DhaK and DhaL homologs (paralogs) associated with putative transcription regulators of the TetR and DeoR families, and proteins with a circularly permuted domain order that belong to the DegV famil

High resolution digital imaging of bacterial cells

The most abundant clone found in ribosomal RNA clone libraries obtained from the world's oceans belongs to the SAR11 phylogenetic group of environmental marine bacteria. Imaging and counting SAR11 bacterial cells in situ has been an important research objective for the past decade. This objective has been especially challenging due to the extremely small size, and hypothetically, the low abundance of ribosomes contained by the cells. To facilitate the imaging of small dim oligotrophic bacterial cells, digital imaging technology featuring very small pixel size, high quantum yield scientific grade CCD chips was integrated with the use of multiple oligonucleotide probes on cells mounted on a non-fluorescing solid substrate. Research into the composition of bacterioplankton populations in natural marine systems follows a two-fold path. Increasing the culturability of microbes found in the natural environment is one research path. Identifying and enumerating the relative fractions of microorganisms in situ by culture-independent methods is another. The accumulation and systematic comparison of ribosomal RNA clones from the marine environment has resulted in a philosophical shift in marine microbiology away from dependence upon cultured strains and toward investigations of in situ molecular signals. The design and use of oligonucleotide DNA probes targeting rRNA targets has matured along with the growth in size and complexity of the public sequence databases. Hybridizing a fluorescently labeled oligonucleotide probe to an rRNA target inside an intact cell provides both phylogenetic and morphological information (a technique called Fluorescence in situ Hybridization (FISH)). To facilitate the imaging of small, dim oligotrophic bacterial cells, digital imaging technology featuring very small pixel size, high quantum yield, scientific grade CCD chips is integrated with the use of multiple oligonucleotide probes on cells mounted on a non-fluorescing solid substrate. This research develops the protocols necessary to acquire and analyze digital images of marine bacterial cells. Experiments were conducted with Bermuda Atlantic Time Series (BATS) environmental samples obtained during cruise BV21 (1998) and B138 (2000). The behavior of the SAR11⁴*Cy3 probe set when hybridized to bacterial cells from these samples was investigated to determine the optimal hybridization reaction conditions. The challenges of bacterial cell counting after cell transfer from PCTE membrane to treated microslides were addressed. Experiments with aged Oregon Coast seawater were performed to investigate the protocol used to transfer cells from membrane to microslides, and examined the distribution of cells and the statistics of counting cells using traditional epifluorescence microscopy and image analysis techniques

Structure of HLA-A*0301 in complex with a peptide of proteolipid protein: insights into the role of HLA-A alleles in susceptibility to multiple sclerosis

The structure of the human major histocompatability (MHC) class I molecule HLA-A*0301 (HLA-A3) in complex with a nonameric peptide (KLIETYFSK) has been determined by X-ray crystallography to 2.7 Å resolution. HLA-A3 is a predisposing allele for multiple sclerosis (MS), an autoimmune disease of the central nervous system. The KLIETYFSK peptide is a naturally processed epitope of proteolipid protein, a myelin protein and candidate target for immune-mediated myelin destruction in MS. Comparison of the structure of HLA-A3 with that of HLA-A2, an MHC class I molecule which is protective against MS, indicates that both MHC class I molecules present very similar faces for T-cell receptor recognition whilst differing in the specificity of their peptide-binding grooves. These characteristics may underlie the opposing (predisposing versus protective) associations that they exhibit both in humans and in mouse models of MS-like disease. Furthermore, subtle alterations within the peptide-binding groove of HLA-A3 and other A3-like MHC class I molecules, members of the so-called A3 superfamily, may be sufficient to alter their presentation of autoantigen peptides such as KLIETYFSK. This in turn may modulate their contribution to the associated risk of autoimmune disease

Automation of large scale transient protein expression in mammalian cells

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI⁻ cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative